WO2019004645A1 - Biosignal measuring bioelectrode based on nanoporous permeable membrane having high specific surface area, and method for manufacturing same - Google Patents

Abstract

The present invention relates to a bioelectrode having improved conductivity, flexibility, and biocompatibility, and a method for manufacturing the same. Particularly, the present invention relates to a conductive polymer bioelectrode comprising a biocompatible polymer substance-based nanoporous permeable membrane having a plurality of pores and an improved surface area, a biosignal transferring pattern layer, and a gold coating layer, based on a PDMS device having low mechanical strength and excellent biocompatibility, whereby the conductive polymer bioelectrode is similar in conductivity to that of a conventional metallic bioelectrode and shows excellent biocompatibility, with the incurrence of a low rejection response. Therefore, it is expected that the conductive polymer bioelectrode of the present invention can replace a metallic bioelectrode, which has poor biosignal transfer efficiency due to the high bioincompatibility thereof.

Description

Porous nanoporous membrane-based bioelectrode for bio signal measurement having high specific surface area and method for manufacturing the same

The present invention relates to a bioelectrode capable of measuring a living body signal based on a porous nanotransflective film having a high specific surface area and having improved capacitance, conductivity, flexibility, and biocompatibility, and a method of manufacturing the same. Specifically, the bioelectrode is a bioelectrode that is inserted into a living body to exchange electrical signals with the body organs and perform electrical interaction with the bioelectrode. The bioelectrode has conductivity similar to that of a conventional metal electrode, but is excellent in flexibility and biocompatibility, There is an advantage that there is no reaction and no immune response.

A bioelectrode is an electrode for measuring and signaling a human body. It is used for the purpose of interacting with tissues and cells by exchanging electrical signals with organs and tissues by attaching them to the body or inserting them into a living body. Because bioelectrodes perform elaborate interactions in the living environment, they require low resistivity, low rejection and good biocompatibility and low mechanical strength to interact with living tissue in order to mediate microscopic electrical signals of living organisms . Conventional metal bioelectrodes have a disadvantage in that an immune response is induced by the inadequate biocompatibility of a metal in contact with living tissues and the efficiency of the bioelectrode is lowered. The bio-incompatibility of the metal means high mechanical strength and ions released from the metal. The biocompatibility of the metal causes the immune cells to gather around the inserted bioelectrode and causes an inflammatory response at the interface of the organ or tissue to which the bioelectrode is attached. The inflammatory reaction hinders signal transmission between the bioelectrode and the organ or tissue and separates the cells from the bioelectrode, so that the resistivity and noise are increased. In addition, conventional metal-based bioelectrodes have mechanical incompatibility due to long-term or tissue movement due to high mechanical strength. The mechanical incompatibility causes a gap between the bioelectrode and the organ or tissue, and the transmission efficiency of the bio-signal due to the gap is reduced. Until now, many disadvantages of the metal-based bioelectrodes have not been solved. Therefore, research on bioelectrodes of new materials has been actively conducted.

The patent documents and references cited herein are hereby incorporated by reference to the same extent as if each reference was individually and clearly identified by reference.

The present inventors have made efforts to develop a variable capacitance bioelectrode based on a porous nano-permeable membrane having a high specific surface area and capable of long-time attachment to a living body surface and implantation in the body for diagnosing and measuring bio-signals. A nanoporous permeable membrane of polyurethane material having excellent biocompatibility and containing a large number of nano-pores is attached to the device (substrate), and patterning for vital signal transmission is performed, followed by gold coating to form a conductive polymer biomolecule electrode . The present inventors have found that the conductive polymer bioelectrode has a low resistivity value at all positions; Porous nanotransflective membranes with high specific surface area ensured by the laminated nanofibers are flexible and have very good conductivity properties and are not deteriorated in mechanical deformation; It is possible to replace the conventional metal-based bioelectrode because the biocompatibility is excellent and the cell can be cultured. Thus, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a conductive polymer bioelectrode comprising a PDMS device, a nanoporous permeable membrane, a patterning for vital signal transmission, and a gold coating layer.

Another object of the present invention is to provide a method for manufacturing the conductive polymer bioelectrode.

Other objects and technical features of the present invention will be described in more detail with reference to the following detailed description, claims and drawings.

According to one aspect of the present invention, there is provided a polydimethylsiloxane (PDMS) device having an intaglio groove to which a nanoporous permeable membrane is attached and having a thickness of 250-350 mu m; A nanoporous permeable membrane having a thickness of 50 to 200 탆 adhered to a depressed groove of the PDMS device; Patterning for transmission of a living body signal formed on the PDMS device and the nanoporous permeable membrane; And a gold (Au) coating layer uniformly formed on the PDMS device, the nanoporous permeable membrane, and the bio-signal transmission patterning layer to a thickness of 0.1-10 탆.

According to an embodiment of the present invention, the nanoporous permeable membrane of the conductive polymer bioelectrode includes a plurality of pores having a diameter of 0.1 to 10 μm, prepared by electrospun biocompatible polymer material to have a thickness of 50 to 200 μm And it is excellent in biocompatibility to enable cell culture.

According to another embodiment of the present invention, the electrospinning is performed under the conditions of a voltage of 10-20 kV, a radiation rate of 0.05-0.3 ml / h, a diameter of the injection needle of 20-30 G, a radiation distance of 20-40 cm, Followed by spinning 4-6 ml of an electrical spinning solution containing the biocompatible polymer substance in an atmosphere at a temperature of 25-35 ° C.

According to another aspect of the present invention, there is provided a method of manufacturing a PDMS device, comprising the steps of: a) preparing a 3D substrate for a PDMS device comprising an intaglio groove for attaching a nanoporous transmissive film; b) a second step of fabricating a PDMS device including an intaglio groove for attaching a nanoporous permeable membrane using the 3D substrate; c) a third step of preparing a nanoporous permeable membrane using electrospinning; d) attaching the nanoporous permeable membrane to the indented groove formed in the PDMS device using a PDMS solution; e) performing a patterning process for transmitting a biological signal to the PDMS device having the nanoporous permeable membrane attached thereto; And f) performing a gold coating on the PDMS device on which the biological signal transmission patterning is performed.

The present invention relates to a bioelectrode having improved conductivity, flexibility, and biocompatibility, and a method of manufacturing the same. More particularly, the present invention relates to a nanoporous permeable membrane based on a PDMS device having a low mechanical strength and excellent biocompatibility based on a biocompatible polymer substance having a large number of voids and having a surface area, The present invention relates to a conductive polymer bioelectrode having conductivity similar to that of a metal material bioelectrode but having a high biocompatibility and a low rejection. Accordingly, the conductive polymer bioelectrode of the present invention is expected to be able to replace the bioelectrode of a metal material, which is deteriorated in bio-signal transmission efficiency due to high bio-incompatibility.

1 shows a view of a 3D substrate and a PDMS device for a PDMS device comprising an intaglio groove for depositing a nanoporous transmissive film. Panel A shows a view of a 3D substrate for a PDMS device with a square shaped nano-porous transmembrane attachment recess. Panel B shows a PDMS device with an indentation groove for various shapes of nano-permeable membrane attachment made from a 3D substrate for a PDMS device.

FIG. 2 shows a 3D substrate for a PDMS device including an angular depression for attaching a square-shaped nanoporous transmission film manufactured using 3D printing.

FIG. 3 shows a PDMS device including a rectangular-shaped nano-porous film for affixed depressions formed by curing a PDMS solution in a 3D substrate for a PDMS device.

Figure 4 shows an optical microscope picture of a PDMS device with a nanoporous permeable membrane. Panel A shows a cross-sectional photograph of a PDMS device with a nanoporous permeable membrane imaged using a 10X optical microscope. Panel B shows a cross-sectional photograph of a PDMS device with a nanoporous permeable membrane imaged using a 50X optical microscope.

FIG. 5 is a photograph of a nano-porous film attached to a PDMS device using a Field Emission Scanning Electron Microscope (FESEM).

FIG. 6 shows a conductive polymer bioelectrode prepared by performing gold coating on a PDMS device having a nanoporous permeable membrane attached thereto. Panel A shows a conductive polymer bioelectrode; Panel B shows a photograph of a gold-coated nanoporous permeable membrane portion of a conductive polymer bioelectrode, taken using FESEM; Panel C shows photographs of the gold coated PDMS device portion of the conductive polymer bioelectrode, taken using FESEM.

7 shows the result of measuring the surface resistivity of the conductive polymer bioelectrode. The surface resistivity was measured five times for the gold coated nanoporous permeable membrane and the gold coated PDMS device, and the average value was calculated.

FIG. 8 shows physical changes of the conductive polymer bioelectrode before and after the mechanical deformation (bending) observed using the FESEM. The bending was performed more than 5 times. The gold coated PDMS device and the nanoporous permeable membrane were photographed with FESEM to observe the gold coating deformation, morphology change of the nanofiber, and pore change.

FIG. 9 shows physical changes of the conductive polymer bioelectrode before and after the mechanical deformation observed using an atomic force microscope (AFM). Panel A shows the results of the gold-coated nanoporous permeable membrane before mechanical deformation, and Panel B shows the results of the gold-coated nanoporous permeable membrane after mechanical deformation.

10 shows the results of observing changes in the nanoporous permeable membrane using FESEM after immersing the conductive polymer bioelectrode in the third distilled water at 36.5 ° C for 0, 7, 14, 21, and 28 days and measuring the surface resistivity of the nanofiltrated membrane Show the results.

FIG. 11 shows the results of observing cell viability using a Live & Dead solution after 5 days of incubation of EpH4-Ras cells and C6 cells in a nanoporous permeable membrane of a conductive bioelectrode.

The present invention relates to a polydimethylsiloxane (PDMS) device having an intaglio groove to which a nanoporous permeable membrane is attached and having a thickness of 250-350 mu m; A nanoporous permeable membrane having a thickness of 50 to 200 탆 adhered to a depressed groove of the PDMS device; Patterning for transmission of a living body signal formed on the PDMS device and the nanoporous permeable membrane; And

And a gold (Au) coating layer uniformly formed on the PDMS device, the nanoporous permeable membrane, and the bio-signal transmission patterning layer to a thickness of 0.1-10 mu m.

According to one aspect of the present invention, there is provided a polydimethylsiloxane (PDMS) device having an intaglio groove to which a nanoporous permeable membrane is attached and having a thickness of 250-350 mu m; A nanoporous permeable membrane having a thickness of 50 to 200 탆 adhered to a depressed groove of the PDMS device; Patterning for transmission of a living body signal formed on the PDMS device and the nanoporous permeable membrane; And a gold coating layer uniformly formed on the PDMS device, the nanoporous permeable membrane, and the bio-signal transmission patterning layer to a thickness of 0.1-10 탆.

The bioelectrode of the present invention refers to an electrode that is inserted into a living body to exchange electrical signals with organs or tissues, or to perform electrical interaction. Since the bioelectrode is inserted into a living body, it is preferable that the biocompatibility is high. The degree of biocompatibility can be determined according to the degree of immunological rejection that occurs when the bioelectrode is inserted. The immunological rejection reaction mainly refers to an inflammatory reaction. Therefore, when the biocompatibility is excellent, the inflammatory reaction due to the insertion of the bioelectrode is reduced. When the inflammatory reaction occurs, the immune cells are collected into organs or tissues to which the bioelectrode is attached, and peripheral cells are killed by the inflammation substances secreted from the immune cells, and the tissue becomes thick. When the tissue is thickened, the resistivity increases, so that the bioelectric signal can not be effectively transmitted. The inflammatory response can be caused by various intracellular substances secreted from the death of the cell. Therefore, there is a method of evaluating the cytotoxicity of the bioelectrode by a method of determining the biocompatibility of the bioelectrode. In the present invention, cell culture using a bioelectrode was attempted to evaluate the biocompatibility of a bioelectrode. The bioelectrode of the present invention includes a nanoporous permeable membrane prepared using a polymer material having excellent biocompatibility. Since the permeable membrane contains nano-sized pores, the cell culture solution can be stored, and cell culture can be performed. The biocompatibility can be evaluated by comparing the number of living cells and the number of dead cells through staining after cell culture. Conventional metal-based bioelectrodes mainly use platinum, platinum alloy or gold. The metal material has good conductivity, but releases excessive metal ions and has a high mechanical strength, which induces an inflammatory reaction, and thus has a disadvantage that the delivery rate of a biological signal decreases over time. According to one embodiment of the present invention, the bioelectrode of the present invention is manufactured by using polydimethylsiloxane (PDMS) rather than a metal material. PDMS is a kind of silicon, which is a polymer material having inactive characteristics and has a low cost, excellent thermal stability and biocompatibility, and thus is widely used in human tissue engineering research. Bioelectrodes attached in vivo may undergo mechanical deformation such as bending or bending due to organ and tissue movements. As described above, the bioelectrode made of a metal material having a high mechanical strength is deformed by the mechanical deformation, or the inflammation reaction (foreign body reaction, foreign body reaction) by the foreign body is induced, Performance may be degraded. On the other hand, the PDMS of the present invention has a merit that the PDMS of the present invention is not deformed by external physical force and maintains its shape because of its low mechanical strength, and can be appropriately processed according to the appearance of organ or tissue, It is possible to improve the transmission efficiency of the antenna. According to an embodiment of the present invention, the PDMS device of the present invention has a thickness of 250-350 탆. Preferably, the PDMS device has a thickness of 275 to 325 占 퐉. More preferably, the thickness of the PDMS device is 300 mu m. When the thickness is 250 탆 or less, separation of the PDMS device from the 3D substrate is difficult due to too high mechanical strength when the PDMS device is manufactured using the 3D substrate. If the thickness exceeds 350 탆, the mechanical strength may increase and the biocompatibility may be deteriorated . The PDMS is low in conductivity since it is used as an electrode as a polymer material. In the present invention, a PDMS-based bioelectrode having excellent conductivity is manufactured by using a nanoporous permeable membrane made of a nanofiber bundle and a gold (Au) coating. To this end, the PDMS device includes an intaglio groove to which the nanoporous permeable membrane can be attached. According to an embodiment of the present invention, the PDMS device has a concave groove with a depth of 100 - 200 탆 for attaching the nanoporous permeable membrane in an area of 25-50% of the total area. Since the nano-porous membrane is attached to the engraved groove, the area of the engraved groove is equal to the area of the nano-porous membrane. If the area of the depressed grooves is less than 25% of the area of the PDMS device, flexibility, surface area, and biocompatibility of the nanoporous permeable membrane are improved, and the area of the depressed grooves is less than 50% %, The mechanical strength is too low and the workability of the bioelectrode may be deteriorated. The nanoporous permeable membrane can be fabricated as a permeable membrane having nano-sized pores formed by stacking polymeric materials having excellent biocompatibility in the form of nanofibers using an electrospinning method. The polymeric material is preferably a material having good moldability, low hardness and mechanical strength, good chemical resistance, no heat resistance, and excellent biocompatibility. According to an embodiment of the present invention, the biocompatible polymer material may be selected from the group consisting of polyurethane, polyacetal, polyamide, polyamide elastomer, polyester, but are not limited to, polyester elastomer, polystyrene, polypropylene, polyacrylonitrile, poly (methymethacrylate), polyolefin, polysulfone, polyvinyl chloride (poly (vinyl chloride)), silicon (silicon), and polyethylene (polyethylene). Preferably, the biocompatible polymer material is polyurethane. The electrospinning method means that the polymer solution having a viscosity is radiated into a fiber form instantaneously using an electrostatic force. When the electrospinning is performed, a laminated membrane in the form of nanofibers can be manufactured. The membrane can have pores depending on the shape of the nanofiber, and the pores can store a physiologically active substance, a drug for cell activation and the like, and research on the material of the nanofiltration drug delivery system is under way. According to an embodiment of the present invention, the nanoporous permeable membrane is a permeable membrane having a thickness of 50-200 탆 produced by electrospinning the biocompatible polymer material and includes a plurality of pores having a diameter of 0.1-10 탆 . Preferably, the nanoporous permeable membrane is a 100-175 μm thick permeable membrane produced by electrospinning the biocompatible polymer material and includes a plurality of pores having a diameter of 0.1-10 μm. More preferably, the nanoporous permeable membrane is a 150 탆 thick permeable membrane prepared by electrospinning the biocompatible polymer material, and includes a plurality of pores having a diameter of 0.1 to 10 탆. The nanoporous permeable membrane is attached to the intaglio groove of the PDMS device. According to an embodiment of the present invention, the concave grooves have a depth of 100-200 탆. Therefore, when the thickness of the nanoporous permeable membrane is less than 50 탆 or more than 200 탆, the height of the PDMS device is not matched with the thickness of the PDMS device.

According to an embodiment of the present invention, the electrospinning is performed under the conditions of a voltage of 10-20 kV, a spinning rate of 0.05-0.3 ml / h, a diameter of the injection needle of 20-30 G, a radiation distance of 20-40 cm, And 4-6 ml of an electric discharge solution containing the biocompatible polymer substance is spun in an atmosphere at a temperature of 25-35 ° C. Preferably, the electrospinning is carried out under conditions of a voltage of 12.5-17.5 kV, a radial velocity of 0.075-0.2 ml / h, a diameter of the injection needle of 22-27 G, a range of 25-35 cm and a humidity of 25-35% Of the biocompatible polymer substance in an atmosphere of 5 ml of the solution. More preferably, the electrospinning is carried out under the conditions of a voltage of 15 kV, a spinning rate of 0.1 ml / h, a diameter of the injection needle of 25 G, and a radiation distance of 30 cm at an atmosphere of 30% Spinning is carried out by spraying 5 ml. If the nanoporous permeable membrane is manufactured outside the conditions of the electrospinning, a permeable membrane having a thickness different from that of the intaglio groove of the PDMS device is manufactured. Therefore, when the PDMS device is attached to the PDMS device, the height is not matched. According to one embodiment of the present invention, the nanoporous permeable membrane of the present invention comprises a plurality of pores having a diameter of 0.1-10 mu m. Therefore, the nanoporous permeable membrane can store the liquid by the capillary phenomenon. The liquid may be a cell culture medium, a buffer solution, a cell active substance in solution, or a cytostatic substance on a solution, and is preferably a cell culture medium. According to one embodiment of the present invention, the nanoporous permeable membrane is capable of cell culture.

In the conductive polymer bioelectrode of the present invention, the PDMS device is located at the lowest layer; The nanoporous permeable membrane having a thickness similar to the depth of the engraved groove is attached to the intaglio groove of the PDMS device so that the PDMS device and the nanoporous permeable membrane are flattened; Patterning for transmitting biological signals is formed on the PDMS device and the nanoporous permeable membrane; A uniform gold coating layer is positioned on the PDMS device, the nanoporous permeable membrane, and the bio signal transmission patterning. The patterning for vital signal transmission may be composed of a plurality of electrodes in accordance with the use of the biomedical electrode. The gold coating layer is formed on the PDMS device, the nanoporous permeable membrane, and the patterning for vital signal transmission, thereby improving the conductivity of the conductive polymer bioelectrode. According to an embodiment of the present invention, the gold coating layer is uniformly formed on the PDMS device, the nanoporous permeable membrane, and the bio-signal transmission patterning layer to a thickness of 0.1-10 mu m. If the thickness of the gold coating layer is less than 0.1 탆, it is difficult to form a coating layer. If the thickness of the gold coating layer exceeds 10 탆, the coating layer may be broken due to mechanical deformation, The pores of the porous permeable membrane may be clogged and the surface area may be reduced.

According to another aspect of the present invention, the present invention provides a method for producing a conductive polymer bioelectrode comprising the steps of:

a) a first step of producing a 3D substrate for a PDMS device comprising an intaglio groove for attaching a nanoporous permeable membrane;

b) a second step of fabricating a PDMS device including an intaglio groove for attaching a nanoporous permeable membrane using the 3D substrate;

c) a third step of preparing a nanoporous permeable membrane using electrospinning;

d) attaching the nanoporous permeable membrane to the indented groove formed in the PDMS device using a PDMS solution;

e) performing a patterning process for transmitting a biological signal to the PDMS device having the nanoporous permeable membrane attached thereto; And

f) performing a gold coating on the PDMS device on which the bio signal transmission patterning is performed;

Step 1 : Nanoporous Permeable membrane  For attachment Grooved groove  Included PDMS For device  3D board manufacturing

According to one embodiment of the present invention, a 3D substrate for a PDMS device of the present invention can be prepared by the following steps:

a) designing a 3D substrate for fabricating a PDMS device having a thickness of 250-350 [micro] m and having an intaglio groove with a depth of 100-200 [mu] m at which a nanoporous permeable membrane can be adhered;

b) preparing an ink for 3D printing by mixing the UV curable plastic and the melting support wax, and then laminating the ink for 3D printing using a 3D printer to manufacture a 3D substrate for a PDMS device;

c) dissolving the support wax by placing the 3D substrate for the PDMS device in a 60-80 DEG C oven for 0.5-2 hours;

d) immersing the 3D substrate for the PDMS device in which the support wax is dissolved in 50-70 캜 cooking oil, followed by ultrasonic cleaning;

e) immersing the 3D substrate for the PDMS device subjected to the ultrasonic cleaning in an EZ rinse solution for 5-20 minutes and washing; And

f) washing and drying the 3D substrate for the PDMS device washed with the EZ rinse solution using distilled water.

According to an embodiment of the present invention, when the support wax is dissolved in an oven of less than 60 ° C in the 3D substrate for the PDMS device in the step c), the dissolution time is increased, and the 3D substrate for the PDMS device is heated to 80 ° C If the support wax is dissolved in an excess oven, there is a disadvantage that the dissolved wax is burnt and the substrate is uneven.

According to another embodiment of the present invention, when the 3D substrate for the PDMS device is immersed in the cooking oil of less than 50 캜 and the ultrasonic cleaning is performed in the step d), it takes more time to remove the dissolved support wax, The removal efficiency of the dissolved support wax is not improved even when ultrasonic cleaning is performed after immersing in a cooking oil exceeding 70 캜.

Step 2 : Nanoporous Permeable membrane  For attachment Grooved groove  Included PDMS Device  Produce

According to one embodiment of the present invention, a PDMS device of the present invention can be manufactured according to the following steps:

a) mixing the PDMS solution and the curing agent in a weight ratio of 10: 0.5 to 10: 2 (PDMS solution: curing agent) and removing bubbles using a desiccator to prepare a PDMS reaction solution;

b) applying the PDMS reaction solution to a 3D substrate for a dried PDMS device and heat treating the PDMS device in a 40-50 ° C oven for 22-26 hours to produce a PDMS device; And

c) removing the PDMS device from the 3D substrate;

According to an embodiment of the present invention, when the PDMS solution and the curing agent are mixed at less than the weight ratio of 10: 0.5 (PDMS solution: curing agent), the time for curing in the 3D substrate for the PDMS device becomes longer and the PDMS solution and the curing agent It is difficult to remove the bubbles by using the desiccator if the mixing ratio exceeds 10: 2 by weight (PDMS solution: curing agent).

According to another embodiment of the present invention, when the PDMS reaction solution is coated on the 3D substrate for the PDMS device and heat treatment is performed in an oven at 40 ° C or less, the PDMS reaction solution is applied to the 3D substrate for the PDMS device Lt; RTI ID = 0.0 > 50 C < / RTI >

Step 3 : Nano-porosity using electrospinning Permeable membrane  Produce

According to one embodiment of the present invention, the nanoporous permeable membrane of the present invention is produced by the electrospinning method through the following steps:

a) adding 10 to 20 parts by weight of polyurethane to 100 parts by weight of a dimethylformamide solution, and mixing the solution for 22 to 26 hours to prepare a polyurethane electrodeposition solution; And

b) The polyurethane electric furnace solution is applied under the conditions of a voltage of 10-20 kV, a spinning rate of 0.05-0.3 ml / h, a diameter of the injection needle of 20-30 G, a radiation distance of 20-40 cm, a humidity of 20-40% Lt; [deg.] ≫ C in a total of 4-6 ml to prepare a nanoporous permeable membrane having a thickness of 150-250 [mu] m.

The diameter of the nanofibers produced by electrospinning is highly dependent on the spinning conditions. In particular, the viscosity of the electrospinning solution is the largest determinant, and a coarse fiber can be produced if the viscosity of the solution is high. Generally, the diameter of the nanofibers is proportional to the square of the concentration of the electrospinning solution. When the nanofibers are laminated to form a fibrous membrane, pores are formed by the gaps between the fibers. Since the size of the void is proportional to the diameter of the nanofiber, when the diameter of the nanofiber is reduced, a gap between the fibers becomes smaller, so that a void having a smaller diameter is formed. When the diameter of the nanofiber is increased, This large pore is formed. The pores are related to the ability to flow across the membrane, and the degree of storage of the solution depends on the size of the pores.

According to an embodiment of the present invention, when polyurethane is added in an amount of less than 10 parts by weight based on 100 parts by weight of the dimethylformamide solution and an electric discharge solution is prepared, Fibers may be formed and the jet may be emitted into a granular form rather than a fiber to form nanopores. When polyurethane is added and mixed in an amount of more than 20 parts by weight based on 100 parts by weight of the dimethylformamide solution to prepare an electrolytic solution, the viscosity of the spinning solution is too high and the nanofibers having a large diameter are radiated The number of pores formed per unit area is reduced and the pore size is increased, so that the amount of the solution that can be stored is reduced.

According to another embodiment of the present invention, the electrospinning is carried out under conditions of a voltage of 10-20 kV, a spinning rate of 0.05-0.3 ml / h, a diameter of an injection needle of 20-30 G, a radiation distance of 20-40 cm , And a total of 4-6 ml is spun in an atmosphere having a humidity of 20-40% and a temperature of 25-35 ° C. Preferably, the electrospinning is carried out under the conditions of a voltage of 15 kV, a spinning rate of 0.1 ml / h, a diameter of the injection needle of 25 G, a spinning distance of 30 cm, an atmosphere of 30% Lt; / RTI > These conditions are the optimum electrospinning conditions for producing a nanoporous permeable membrane having a plurality of voids with a thickness of 150-250 μm and a diameter of 0.1-10 μm.

Step 4 : PDMS To device  Nanoporous Permeable membrane  Attaching step

The nanoporous permeable membrane prepared above is attached to the PDMS device manufactured in the above step. To this end, a PDMS solution is prepared, and only a small amount of the PDMS solution is applied to the depressed groove of the PDMS device, and then the nanoporous permeable membrane is attached. When an excessive amount of the PDMS solution is used, the PDMS solution is absorbed by the nanoporous permeable membrane due to capillary phenomenon and hardened, so that voids of the permeable membrane disappear. Therefore, only a very small amount of the PDMS solution is used.

Fifth - Step 6 : For vital signal transmission Patterning  And Gold coating

According to an embodiment of the present invention, an electrode is provided so that an electrical signal for transferring the biological signal to the living body or an electrical signal transmitted from the living body can be moved by performing patterning for the biological signal on the PDMS device having the nanoporous permeable membrane attached thereto.

According to another embodiment of the present invention, when patterning for bio-signal transmission is completed, a uniform gold coating layer is formed on the PDMS device, the nanoporous permeable membrane, and the bio-signal transmission patterning layer to a thickness of 0.1-10 μm. The gold coating layer is made of gold (Au) having excellent biocompatibility. Since the gold coating layer is uniformly formed with a thickness of 0.1-10 탆, it is possible to maintain the voids of the nano-porous permeable membrane without blocking while improving conductivity.

Example

Example  One) PDMS device  Fabrication of 3D substrate for fabrication

In order to fabricate a PDMS (Polydimethylsiloxane) device, a 3D substrate for PDMS device fabrication was designed using a 3D design program Inventor (Autodesk, USA) (Fig. 1). The overall shape of the 3D substrate for fabricating the PDMS device is designed in various shapes such as a square, a triangle, a circle, and a pentagon. The 3D substrate for fabricating the PDMS device includes a PDMS device forming unit (embossed shape) in which a PDMS device is formed when the PDMS device is manufactured by pouring a PDMS solution onto the 3D substrate, and a nano-porous And an intaglio groove forming portion for attaching a permeable membrane (see Panel A in Fig. 1). 1, a rectangular PDMS device 3D substrate to which the polyurethane nanoporous transmission layer of Panel A is attached may be formed with a PDMS device having a thickness of 250-350 mu m and a width of 5 cm and a length of 5 cm, A negative electrode groove for attaching a nano-porous porous membrane having a depression shape with a depth of 100 to 200 mu m and a length of 2 to 2.5 cm and a length of 2 to 2.5 cm may be formed. The 3D substrate for PDMS device fabrication was manufactured by using a 3D printer (PROJET 3510 HD) with photocurable principle and VISIJET M3 crystal, which is one kind of ultraviolet (UV) curing plastic, was used as a main material. , Support VISIJET S300, one of the wax materials having both fusibility and non-toxicity, was used. 1, a VISIJET M3 crystal solution was laminated according to the design of Panel A to prepare a 3D substrate for PDMS device fabrication. In order to remove the Support VISIJET S300, an incubation was performed in an oven at 70 ° C for 1 hour. The 3D substrate for PDMS device fabrication, in which Support VISIJET S300 was dissolved through the incubation, was immersed in an ultrasonic washing machine filled with cooking oil at 60 ° C for 1 hour, and then subjected to a first washing process, followed by soaking in EZ Rinse solution for 10 minutes Was carried out. The 3D substrate for PDMS device fabrication, in which the primary and secondary cleaning processes were sequentially performed, was subjected to final tertiary cleaning using distilled water and then dried. Figure 2 shows a 3D substrate for fabricating a PDMS device fabricated by the above method. The 3D substrate for fabricating the PDMS device comprises a PDMS device forming unit for forming a PDMS device, which is a depressed portion, and a depressed groove forming unit for attaching a nano-porous permeable film, to which a polyurethane nanoporous permeable membrane is attached.

Example  2) PDMS Device  Produce

A PDMS reaction solution was prepared to prepare the PDMS device of the present invention. In the PDMS reaction solution, the PDMS solution and the curing agent were mixed at a ratio of 10: 1, and the bubbles were sufficiently removed using a desiccator. 3 ml of the PDMS reaction solution in which the bubbles were removed was coated on the 3D substrate for fabricating the PDMS device, and then heat-treated in a 45 ° C laboratory oven for 24 hours to cure. The cured PDMS device was detached from the 3D substrate for PDMS device fabrication. Fig. 3 shows a PDMS device manufactured by the above method. The PDMS device comprises a PDMS substrate portion and an intaglio groove for attaching a nano-porous permeable membrane to which a polyurethane nanoporous permeable membrane is bonded. The PDMS device has a thickness of 250-350 占 퐉 and a length and a length of 5 cm. The intaglio grooves for attaching the nanoporous permeable membrane in the PDMS device have a depth of 100 - 200 μm and a length of 2 - 2.5 cm.

Example  3) Polyurethane nanoporous Permeable membrane  Produce

The present invention attaches nanofibers to the PDMS device to produce a bioelectrode having improved conductivity and biocompatibility. The polymer material for the preparation of the nanofibers was selected from polyurethane having excellent biocompatibility and a polyurethane nanoporous permeable membrane was prepared by electrospinning. To this solution, 15 parts by weight of polyurethane was added to 100 parts by weight of a dimethylformamide solution and mixed for 24 hours to prepare a polyurethane electrodeposition solution. Voltage 15kV; Spinning rate 0.1 ml / h; Diameter of injection needle 25G; Radiation condition of radiation distance of 30cm; And humidity 30%; And a temperature of 30 ° C .; a polyurethane nanoporous permeable membrane was prepared by electrospinning a total of 5 ml of polyurethane electrodeposition solution. The thickness of the polyurethane nanoporous permeable membrane prepared in the electrospinning condition was similar to the depth (100 ~ 200 um) of the concave groove for attachment of the nanoporous permeable membrane. The thickness of the polyurethane nanoporous permeable membrane (150 to 250 mu m) is judged to be an appropriate thickness considering the intaglio grooves for attaching the nanoporous permeable membrane. In order to prepare the permeable membrane of the above thickness by electrospinning, It is judged that it is preferable to use the amount of use.

Example  4) Polyurethane nanoporous Permeable membrane  Attach

The prepared polyurethane nanoporous permeable membrane was cut so that the length and width of the polyurethane nanoporous permeable membrane were 2 to 2.5 cm so as to fit the intaglio groove for attachment of the nanoporous permeable membrane, and then the uncured pure PDMS solution was spread on the permeable membrane attachment portion, A polyurethane nanoporous permeable membrane was attached. The PDMS device (PU-PDMS device) with the polyurethane nanoporous permeable membrane was placed in a 45 ° C laboratory oven and cured for 1 hour. Figure 4 shows an optical micrograph of a polyurethane nanoporous permeable membrane attached to a PDMS device. As a result, it was confirmed that the polyurethane nanoporous permeable membrane was attached to the PDMS device at a proper thickness. When a large amount of the uncured pure PDMS solution is used at the time of attachment, the PDMS solution is diffused into the pores of the permeable membrane and cured to seal the pores, so that only a very small amount of the PDMS solution is not spread. FIG. 5 shows a field emission scanning electron microscope (FESEM) observation result for a PDMS device to which a polyurethane nanoporous permeable membrane has been successfully attached. According to FIG. 5, the PU-PDMS device correctly adheres the PDMS solution without being diffused into the polyurethane nanoporous permeable membrane, thereby maintaining the porosity of the nanoporous permeable membrane.

Example  5) Fabrication of conductive polymer biomedical electrode

A gold (Au) coating was applied to the PU-PDMS device to prepare a conductive polymeric bioelectrode having conductivity. Gold is safe for human body because it has excellent biocompatibility and non-toxic properties. After conducting patterning to transmit the vital signal, a conducting polymeric bioelectrode capable of transmitting an excellent signal was fabricated by coating gold with 0.1-10 μm thickness. Panel A of FIG. 6 shows a conductive polymer bioelectrode (gold-coated PU-PDMS device) of the present invention. It can be confirmed that the gold coating is thin and the coating layer is uniformly formed on the surface. 6 shows the result of FESEM observation of the gold-coated polyurethane nanoporous permeable membrane portion of the conductive polymer bioelectrode. According to panel B of FIG. 6, it was confirmed that the polyurethane nanoporous permeable membrane was well coated with gold, but the shape and porosity of the nanofiber were well maintained. Panel C of FIG. 6 shows the result of FESEM observation of the PDMS device portion of the conductive polymer bioelectrode. It was confirmed that the PDMS portion of the conductive polymer bioelectrode was uniformly coated with gold as in the PU nanoporous permeable membrane portion of the conductive polymer bioelectrode.

Experimental Example  1) Conductivity of conductive polymer bioelectrode

In order to evaluate the conductivity of the conductive polymer bioelectrode of the present invention, the surface resistivity of the nanoporous permeable membrane portion and the PDMS device portion was measured using a surface resistance meter. FIG. 7 shows the results of measuring the surface resistivity of the nanoporous permeable membrane part and the PDMS device part of the conductive polymer bioelectrode. The PU nanoporous permeable membrane was found to have an average resistivity of 0.157? As a result of measuring the resistivity at six different positions; As a result of measuring the resistivity at five different positions in the PDMS device, it was confirmed that it had an average resistivity of 0.067?; It has been confirmed that the conductive polymer has excellent conductivity at all portions of the bioelectrode.

Experimental Example  2) Durability against physical deformation of conductive polymer bioelectrode

The bioelectrode is injected into the body in various forms. Therefore, the bioelectrode to be inserted into the living body should be free from changes in the porosity of the PDMS device gold coating, the porosity of the gold-coated permeable membrane, and the continuity of the nanofibers even under the application of mechanical pressure. In order to confirm this, the surface of the conductive polymeric bioelectrode and the conducting polymeric bioelectrode, which were bent several times, were observed using an FESEM and an atomic force microscope (AFM). FIG. 8 shows the results of observing the surface state of the conducting polymeric bioelectrode, which has not undergone the bending process (mechanical deformation) using the FESEM, and the conducting polymeric bioelectrode, which has been subjected to the bending process 5 to 7 times. Experimental results showed that the morphology of the nanoporous permeable membrane and the gold coating state did not change. FIG. 9 shows the result of comparing the surface states of the conductive polymer bioelectrode, which has not undergone the bending process, and the conductive polymer bioelectrode, which has undergone the bending process several times, using an atomic force microscope (AFM) . As a result of FESEM, it was confirmed that there was no change in the morphology and gold coating level of the nanoporous permeable membrane, and the PDMS device part was also found to have no change in morphology and gold coating level.

Experimental Example  3) Conductive polymeric bioelectrode In vivo  Durability to the environment

The human body consists of more than 70% water and maintains a constant temperature of 36.5 ℃. Therefore, in order to confirm the durability of the conductive polymer bioelectrode according to the present invention, the physical properties and conductivity of the electrode were observed with time after immersing the bioelectrode in the third distilled water maintained at a temperature of 36.5 ° C.

10 is a graph showing the results of measurement of the permeability of the nanoporous permeable membrane of the present invention by using a scanning electron microscope (SEM) after immersing the conductive polymer bioelectrode of the present invention in the third distilled water maintained at 36.5 ° C for 7 days, 14 days, 21 days, Shows the result of shooting the shape. As a result of the experiment, it was found that the nanoporous permeable membrane of the conductive polymer bioelectrode immersed in the third distilled water for 28 days has the following properties: the state of the nanoporous permeable membrane and the gold coating layer not immersed in the third distilled water, the shape of the nanofiber, There was no difference.

To confirm that the conductivity of the conductive polymer bioelectrode was maintained in vivo, the resistivity of the surface was measured by immersion under the same conditions as described above (see FIG. 10). As a result of the measurement, it was confirmed that the immersed conductive polymer bioelectrode had a resistance value of 0.31-0.84? And an average resistance value of 0.59 ?. Therefore, the conductive polymer bioelectrode was similar to the first resistance value of 0.58? Despite being immersed in the third distilled water maintained at a temperature of 36.5 占 폚 for 28 days.

Experimental Example  4) Biocompatibility of conductive polymer bioelectrode

To confirm the biocompatibility of the conductive polymer bioelectrode, EpH4-Ras cells treated with cancer gene Ras and C6 cells, one type of fibroblasts, were cultured in rat epithelial cells on a nano-porous membrane for 5 days, and Live / Dead solution (The LIVE / DEAD ( ^R) Cell Imaging Kit (Thermo Fisher Scientific)) to determine cell viability (see FIG. 11). By using the Live / Dead solution, it is possible to distinguish living cells that retain cell membranes from dead cells that do not retain cell membranes. When the live / dead solution was added to the cultured cells and observed using a fluorescence microscope equipped with FITC and Texas RED filters, living cells were stained with a whole green fluorescence (FITC, ex / em = 488 nm / 515 nm) Cells are stained with red fluorescence (Texas RED, ex / em = 570nm / 602nm) by exposed DNA due to nuclear decay. Experimental results show that the EpH4-Ras cells or C6 cells cultured on the nanoporous permeable membrane have survived to a higher number of living cells than the number of dead cells (see FIG. 11). Therefore, it has been confirmed that the conductive polymer bioelectrode of the present invention has excellent biocompatibility to enable cell culture.

The specific embodiments described herein are representative of preferred embodiments or examples of the present invention, and thus the scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that modifications and other uses of the invention do not depart from the scope of the invention described in the claims.

The present invention relates to a bioelectrode for bio signal measurement based on a porous nanofiltration membrane having a high specific surface area and a method for manufacturing the bioelectrode and a method for manufacturing the bioelectrode, which can replace a metal electrode of a metal material, .

Claims (10)

1. A polydimethylsiloxane (PDMS) device having an intaglio groove with a nanoporous permeable membrane and having a thickness of 250-350 占 퐉;

   A nanoporous permeable membrane having a thickness of 50 to 200 탆 adhered to a depressed groove of the PDMS device;

   Patterning for transmission of a living body signal formed on the PDMS device and the nanoporous permeable membrane; And

   A gold (Au) coating layer uniformly formed on the PDMS device, the nanoporous permeable membrane, and the bio-signal transmission patterning to a thickness of 0.1-10 mu m;

   And a conductive polymer.
2. The conductive polymer biomolecule according to claim 1, wherein the PDMS device is an intaglio groove having a depth of 100 to 200 mu m for attaching the nanoporous permeable membrane.
3. The nanoporous permeable membrane according to claim 1, wherein the nanoporous permeable membrane is a permeable membrane having a thickness of 50 to 200 탆 produced by electrospinning a biocompatible polymer material and includes a plurality of pores having a diameter of 0.1 to 10 탆 Wherein the conductive polymer is a biomolecule.
4. The biocompatible polymeric material of claim 3, wherein the biocompatible polymeric material is selected from the group consisting of polyurethane, polyacetal, polyamide, polyamide elastomer, polyester, polyester elastomer, ), Polystyrene, polypropylene, polyacrylonitrile, poly (methymethacrylate), polyolefin, polysulfone, poly (vinyl chloride) vinyl chloride, silicon, and polyethylene. 2. The conductive polymer biomolecule according to claim 1,
5. 4. The method according to claim 3, wherein the electrospinning is performed under the conditions of a voltage of 10-20 kV, a spinning rate of 0.05-0.3 ml / h, a diameter of the injection needle of 20-30 G and a radiation distance of 20-40 cm, And 4-6 ml of an electric discharge solution containing the biocompatible polymer substance is spun in an atmosphere at 35 캜.
6. The conductive polymer biomolecule according to claim 1, wherein the nanoporous permeable membrane is capable of cell culture.
7. a) a first step of producing a 3D substrate for a PDMS device comprising an intaglio groove for attaching a nanoporous permeable membrane;

   b) a second step of fabricating a PDMS device including an intaglio groove for attaching a nanoporous permeable membrane using the 3D substrate;

   c) a third step of preparing a nanoporous permeable membrane using electrospinning;

   d) attaching the nanoporous permeable membrane to the indented groove formed in the PDMS device using a PDMS solution;

   e) performing a patterning process for transmitting a biological signal to the PDMS device having the nanoporous permeable membrane attached thereto; And

   f) performing a gold coating on the PDMS device on which the bio signal transmission patterning is performed;

   Wherein the conductive polymer is a polymer electrolyte.
8. 8. The method of claim 7, wherein the first step

   a) designing a 3D substrate for fabricating a PDMS device having a thickness of 250-350 占 퐉 and having an intaglio groove with a depth of 100-200 占 퐉 to which a nanoporous permeable membrane for cell culture can be adhered;

   b) preparing an ink for 3D printing by mixing the UV curable plastic and the melting support wax, and then laminating the ink for 3D printing using a 3D printer to manufacture a 3D substrate for a PDMS device;

   c) dissolving the support wax by placing the 3D substrate for the PDMS device in a 60-80 DEG C oven for 0.5-2 hours;

   d) immersing the 3D substrate for the PDMS device in which the support wax is dissolved in 50-70 캜 cooking oil, followed by ultrasonic cleaning;

   e) immersing the 3D substrate for the PDMS device subjected to the ultrasonic cleaning in an EZ rinse solution for 5-20 minutes and washing; And

   f) washing and drying the 3D substrate for the PDMS device cleaned with the EZ rinse solution using distilled water;

   Wherein the polymer electrolyte membrane comprises a polymer electrolyte.
9. 8. The method of claim 7, wherein the second step comprises:

   a) mixing the PDMS solution and the curing agent in a weight ratio of 10: 0.5 to 10: 2 (PDMS solution: curing agent) and removing bubbles using a desiccator to prepare a PDMS reaction solution;

   b) applying the PDMS reaction solution to a 3D substrate for a dried PDMS device and heat treating the PDMS device in a 40-50 ° C oven for 22-26 hours to produce a PDMS device; And

   c) removing the PDMS device from the 3D substrate;

   Wherein the polymer electrolyte membrane comprises a polymer electrolyte.
10. 8. The method of claim 7, wherein the third step comprises:

    a) adding 10 to 20 parts by weight of polyurethane to 100 parts by weight of a dimethylformamide solution, and mixing the solution for 22 to 26 hours to prepare a polyurethane electrodeposition solution; And

    b) The polyurethane electrospinning solution was applied under the conditions of a voltage of 10-20 kV, a spinning rate of 0.05-0.3 ml / h, a diameter of the injection needle of 20-30 G, a spinning distance of 20-40 cm, a humidity of 20-40% Spinning a total of 4-6 ml in an atmosphere at 35 캜 to produce a nanoporous permeable membrane having a thickness of 150-250 탆;

    Wherein the polymer electrolyte membrane comprises a polymer electrolyte.

Images